Feline immunodeficiency virus (FIV) is an exogenous retrovirus of the Lentivirus genus and is associated with a fatal acquired immunodeficiency syndrome (AIDS)-like disease in domestic cats. FIV is similar in genetic organization and virion morphology to other members of the genus lentivirinae, including: human immunodeficiency virus types 1 and 2 (HIV-1, HIV-2), simian immunodeficiency virus (SIV), visna-maedi virus (VMV), equine infectious anemia virus (EIAV), caprine arthritis-encephalitis virus (CAEV), and bovine immunodeficiency-like virus (BIV) {Clements, J. E., et al., Seminars in Virology, 3:137-146 (1992); and Shacklett, B. L., et al., Virol., 204:860-867 (1994)}. Both HIV and FIV cause a fatal syndrome in their respective hosts. This syndrome is characterized by generalized lymphadenopathy and increased susceptibility to opportunistic infections.
Biological FIV-PPR and isolates of molecular clones of FIV-pF34 (the molecular proviral clone of the latter is termed pF34) are described in Talbott, R. L., et al., PNAS (USA), 86:5743-5747 (1989). The molecular clone FIV-pF34 infected Crandell feline kidney (CrFK) and G355-5 cell lines, but replicated less efficiently in feline peripheral blood leukocytes (Id.). In contrast, the PPR clone productively infected the primary feline peripheral blood leukocytes but not CrFK or G355-5 cells (Id.). The isolate of another molecular clone, FIV-pPPR (the molecular proviral clone is termed pPPR) was reported in Phillips, T. R., et al., J. Vir., 64(10):4605-4613 (1990). The two viral isolates (pPPR and pF34) show more than 85% sequence homology {Sparger, E. E., et al., Virol., 187:165-177 (1992)}.
Lentiviruses have complex genomes which encode structural proteins (e.g., Gag, Pol, and Env) as well as regulatory proteins (e.g., Tat, Rev) and accessory proteins (e.g., Vif, Nef, Vpr, Vpx, Vpu) (Id). FIV has been shown to encode a rev gene and a vif gene, but appears to lack genes corresponding to tat, nef, vpr, vpx, and vpu (Id).
Tomonaga, et al., reported that mutation of a short conserved region designated open reading frame A (ORF-A) in FIV clone TM-2 produced a virus that replicated with delayed kinetics in feline lymphoid cell lines and peripheral blood lymphocytes (PBL) {Tomanaga, K., et al., J. Virol., 67:5889-5895 (1993)}. Shacklett, et al., supra, made three mutations in the vif gene of molecular clone FIV-pF34: (i) deletion of 223 bp from the central portion of the gene; (ii) site-directed mutation of a conserved N-terminal basic region; and (iii) site-directed mutation of a conserved C-terminal motif. FIV proviruses containing each of these mutations were tested for replication following transfection into two feline adherent cell types, CrFK and G355-5. All three vif mutants produced very little cell-free virus or viral protein in both cell types (Id.).
The long terminal repeat (LTR) of a retrovirus contains sequence elements that constitute a promoter for controlling viral gene expression in infected cells. The FIV LTR was found to contain an element (i.e., a potential AP-1 site) upstream from the TATA box which was required for responses to T-cell activation signals. In addition, transcription directed by the LTR responded to an inducer of intracellular cyclic-AMP (c-AMP) (i.e., forskolin). Mutagenesis studies revealed that a potential ATF site, also known as a c-AMP response element (CRE) is required for activation by either forskolin or dibutyryl c-AMP.
FIV LTR mutations affecting the first AP4 site, AP1 site, ATF site, or NF-KB site resulted in decreased basal promoter activity of LTR as measured in various cell lines in transient expression assays using plasmids containing the viral LTR linked to the bacterial chloramphenicol acetyltransferase gene {Sparger, E. E., et al., Virol., 187:165-177 (1992)}. Miyazawa, T., et al., deleted sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of FIV LTR {Miyazawa, T., et al., J. Gen. Virol., 74:1573-1580 (1993)}. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild type in two feline CD4-positive T lymphoblastoid cell lines. Miyazawa, T., et al., found that the rate and activity of the mutant were almost the same as those of the wild type. Miyazawa, T., et al., concluded that the 31 bp fragment was important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.
It has been long recognized that DNA of molecularly cloned DNA viruses can be highly infectious in vivo, but the infectious nature of retroviral DNA in vivo has not been generally appreciated. See, for example, the disclosures of U.S. Pat. Nos. 5,589,466 and 5,152,982. However, Myrick, K. V., et al., found that intact SIVmac could be isolated from peripheral blood lymphocytes of three of four Macaca fascicularis monkeys which were inoculated, intramuscularly, with SIVmac proviral DNA. Letvin, N. L., et al., Nature, 349:573 (1991). Infectious virus was also detected in the spleens of mice after injection with cloned chimeric murine retroviral DNA of FrCas.sup.E. Portis, J. L., et al., J. Acquired Immune Deficiency Syndrome, 5:1272-1277 (1992). A plasmid containing an unpermuted genome of FB29, flanked by two LTRs, was infectious when injected as supercoiled DNA (without excision of the viral genome). Id.
Ulmer, J. B., et al., injected plasmid DNA encoding influenza A nucleoprotein into the quadriceps of BALB/c mice. This resulted in the generation of nucleoprotein-specific cytotoxic T-lymphocytes and protection from a subsequent challenge with a heterologous strain of influenza A virus, as measured by decreased viral lung titers, inhibition of mass loss, and increased survival. Ulmer, J. B., et al., Sci., 259:1745-1749 (1993). RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and .beta.-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. Wolff, J. A., et al., Sci., 247:1465-1468 (1990).
The preparation of vaccines to protect feline hosts against FIV infection is discussed in U.S. Pat. Nos. 5,275,813 and 5,510,106 and in Hosie (1994) Br. Vet. J. 150:25-39.